Temperature-mediated dynamics: Unravelling the impact of temperature on cuticular hydrocarbon profiles, mating behaviour, and life history traits in three Drosophila species

In a world grappling with climate change, understanding the enduring impact of changes in temperatures on insect adult traits is crucial. It is proposed that cold- and warm-adapted species exhibit specialized behavioural and physiological responses to their respective temperature ranges. In contrast, generalist species maintain more stable metabolic and developmental rates across a broader range of temperatures, reflecting their ability to exploit diverse thermal niches. Here, we explored this intricate response to temperature exposure in three Drosophila species: Drosophila ezoana originating in Arctic regions, D. novamexicana in arid, hot environments, and in the cosmopolitan species D. virilis. Rearing these flies at 15, 20, 25, and 30 °C revealed striking variations in their cuticular hydrocarbon (CHC) profiles, known to mediate mate recognition and prevent water loss in insects. The cold-adapted D. ezoana consistently exhibited reduced CHC levels with increasing temperatures, while the warm-adapted D. novamexicana and the cosmopolitan D. virilis displayed more nuanced responses. Additionally, we observed a significant influence of rearing temperature on the mating behaviour of these flies, where those reared at the extreme temperatures, 15 and 30 °C, exhibiting reduced mating success. Consequently, this led to a decrease in the production of adult offspring. Also, these adult offspring underwent notable alterations in life history traits, reaching adulthood more rapidly at 25 and 30 °C but with lower weight and reduced longevity. Furthermore, among these offspring, those produced by the cold-adapted D. ezoana were more vulnerable to desiccation and starvation than those from the warm-adapted D. novamexicana and the cosmopolitan D. virilis. In summary, our research demonstrates that Drosophila species from diverse ecological regions exhibit distinct responses to temperature changes, as evidenced by variations in CHC profiles, mating behaviours, fertility, and life history traits. This provides valuable insights into how environmental conditions shape the biology and ecology of insects.


Introduction
Butterflies, bees, moths, beetles, and flies all display a complete metamorphosis (holometaboly) as they progress through their developmental stages before reaching adulthood [1].In these insects, the immature stages (eggs, larvae and pupae) have no physical resemblance to the adult stages and undergo substantial morphological and physiological transformations to complete their development.In this process, the hatching egg produces a larva that undergoes multiple moults (or ecdysis), during which the old cuticle is shed and replaced with a new one.This transformation ultimately leads to the formation of a pupa, from which an adult will eventually emerge [2].During this transformation, a significant restructuring occurs in larval tissues and organs, involving the nervous system, midgut, muscles and cuticle [3].Going these changes can impose considerable stress and challenges for the insects.Any environmental conditions that are inconsistent or unfavourable can further intensify this stress, potentially impeding the success of the transformation process.A varying environment often triggers plasticity that generates multiple phenotypes in insects with the same genome [4].
Among insect organs, the cuticle is highly susceptible to environmental changes due to its direct exposure to the external world.Within this organ, specialized cells called oenocytes secrete cuticular hydrocarbons [5], which often serve a dual role [6,7].They protect terrestrial insects against desiccation.For example, using 50 drosophilid species, Wang et al. [8] demonstrated that the composition of cuticular hydrocarbons (CHCs) can explain up to 85.5 % of the variability in desiccation resistance.Also, CHCs serve as signalling molecules in a wide variety of chemical communication systems.They serve as the primary signals to attract conspecific mates for courtship and copulation behaviours, as well as to indicate receptivity, fertility, and mating status [9,10].The production of cuticular hydrocarbons in adult insects can be influenced by the stress experienced during preimaginal development.For example, in Drosophila mojavensis, adults reared on their natural host (cactus) produced more CHCs as opposed to those reared on laboratory food [11].Eggs of D. melanogaster ([12,13]) and D. mojavensis [14] incubated under different temperature conditions give rise to adults with different CHC profiles.However, the extent to which these changes in CHC profiles can impact the mating behaviour of insects and the fitness of their subsequent offspring remains uncertain.
The current climate changes, primarily driven by human activities, are causing a rise in global temperatures [9,15].As a consequence, insects are encountering temperatures beyond what has been present in their historical ranges [16,17].Cold-adapted species exhibit narrow thermal tolerance ranges centered around lower temperatures, with lower optimal temperatures for growth, reproduction, and metabolism, and are equipped with physiological mechanisms to cope with freezing conditions [18,19].Warm-adapted species, conversely, have narrow tolerance ranges centered on higher temperatures, higher optimal temperatures, and mechanisms for efficient heat dissipation [20,21].Generalist species display broad thermal tolerance ranges, intermediate or flexible optimal temperatures, and versatile physiological adaptations, allowing them to thrive across diverse thermal environments [22,23].Consequently, cold-and warm-adapted species have specialized behavioral and physiological responses to their respective temperature ranges, whereas generalists maintain more stable metabolic and developmental rates across a wider range of temperatures, reflecting their ability to exploit various thermal niches [24,25].It is postulated that holometabolous insects may exhibit variable thermal adaptations and responses to suboptimal temperatures based on their geographical distribution [26][27][28].As reviewed by Hodkinson [29], this adaptation to local climate conditions can happen through shifts in insect behaviour (e.g.mating frequency and duration), fertility, life history traits (e.g.developmental time, body size and longevity) and physiological attributes (e.g.desiccation and starvation resistance).Here, we aimed to investigate how rising rearing temperatures affect the reproduction, fitness, and physiology of insects living in different latitudinal zones.We hypothesized that the composition of the CHC layer in drosophilid species from varying latitudes would change with increasing developmental temperatures.Furthermore, these temperature changes could significantly influence their mating behaviour, fertility, various life history traits (such as developmental time, body size, and longevity), as well as the desiccation and starvation resistance of their offspring.To test our hypothesis, we selected three closely related species within the virilis group: Drosophila ezoana (Holarctic distribution, e.g.Finland), D. novamexicana (Neotropical distribution, e.g.Mexico) and D. virilis (Cosmopolitan distribution).These species have adapted to various climates, particularly different temperature ranges, and display distinct temperature preferences [30].
We analysed the cuticular hydrocarbon compositions of adults of the three drosophilid species when developed at 15, 20, 25, and 30 • C. Next, we investigated whether these flies exhibit distinct mating behaviour depending on rearing temperature.Also, we assessed their fertility and determined the life history traits of their offspring.Finally, we tested how well the offspring could endure extended periods of extreme dryness (desiccation) and food deprivation (starvation).Our study provides comprehensive insights into how insects from various latitudinal regions are likely to respond to the ongoing global warming.

Flies' preparation and cuticular hydrocarbon analysis
Gravid females of D. ezoana, D. novamexicana, and D. virilis from the stocks were allowed to lay eggs in food vials at low density (20-30) for 5 h.These vials were immediately transferred inside four Snider Scientific incubators (www.snijderslabs.com)set respectively at 15, 20, 25 and 30 • C, all with 70 % humidity and 12 h Light:12 h Dark.Once emerged, flies were collected within 1 h, sexed and returned to their respective developmental incubator.Seven days later, 5 flies of the same sex from each incubator were soaked for 10 min in 200 μl hexane (Fig. 1) containing 1-Bromoeicosane (25 ng/μl) as an internal standard [8,31].Each body extract sample was analysed by gas chromatography coupled with mass spectrometry (7890B GC System, 5977A MSD, Agilent Technologies, https://www.agilent.com)equipped with a polar column (HP-INNOWAX, 30 m long, 0.25 mm inner diameter, 25 μm film thickness; Agilent) with helium as carrier gas.Each sample (1 μL) was injected into the GC-MS with an autosampler (Agilent Technologies).The inlet temperature was set to 250 • C. The temperature of the GC oven was held at 50 • C for 2 min, increased gradually (15 • C/min) to 250 • C, which was held for 3 min, and then to 280 • C (20 • C/min) and held for 30 min.The MS transfer line, source, and quad were held at 280, 230, and 150 • C, respectively.GC-MS data were processed with the MDS-ChemStation Enhanced Data Analysis software (Agilent).We identified each CHC using the library from the National Institute of Standards & Technology (NIST).For each developmental temperature and species, we injected 5 samples per sex.In every sample, we determined the quantity of each identified CHC by comparing its peak area with that of the internal standard (normalized intensity).

Mating assay
To investigate the impact of temperature on the mating behaviour of D. ezoana, D. novamexicana, and D. virilis, virgin individuals of these flies reared at 15, 20, 25, and 30 • C were separated by sex upon emergence, placed in groups (20 individuals per vial), and returned to their respective incubator.Seven days later, a single male and a single female from the same temperature treatment were introduced (using a mouth aspirator) in a courtship chamber and their behaviour was observed for 1 h (Fig. 1).Each courtship arena was equipped with 4 chambers (1 cm diameter × 0.5 cm depth) covered with a plastic slide.A constant airflow of 0.2 mL/min was added from below to each arena.To record the courtship behaviours of the couples, the four chambers were observed simultaneously using a GoPro Camera 4 or Logitech C615.For each developmental temperature, we observed 48 couples.To quantify the mating behaviour of each pair, we manually examined each video to determine mating success (i.e., the percentage of males that managed to mount the females) and mating duration (i.e., the amount of time a male spent on top of the female).All these mating assays were performed at 23 • C and 70 % humidity.

Fertility assay
To elucidate how temperature impacts the fertility of D. ezoana, D. novamexicana, and D. virilis, each mating pair that managed to mate in the previous experiment was gently collected (using a mouth aspirator) and placed in a vial containing fresh food (13 g) and transferred to their corresponding incubator (previously set at 15, 20, 25, or 30 • C).For each couple, we allowed the female to lay eggs throughout its lifespan and closely observed the progress of egg development until the emergence of adult offspring.This allowed us to count the total number of adult offspring (fertility) produced by each species at different developmental temperatures.Each mating pair was transferred to a new food vial each day.

Determination of life history traits
During the fertility test, we simultaneously collected data on developmental duration (from egg to adult), pupal mass, and adult body mass (both fresh and dry) in resulting adult offspring.For each experimental temperature, we measured the body mass of 30 pupae/species individually using a Sartorius balance (0.0001 g precision).To determine the fresh adult body mass of each species, we placed 10 males and 10 females from each temperature group into pre-weighed 1.5 mL Eppendorf tubes and recorded the total body mass using a Sartorius balance.For each developmental temperature, species, and sex, 10 independent trials were conducted.To measure the dry body mass in each trial, we took these tubes, placed them in an oven set at 40 • C for 10 min to kill the flies, and then transferred them to room temperature.We subsequently weighed them every two days until a constant mass was noted.The fresh and dry body mass was obtained by subtracting the mass of the Eppendorf tube from the mass of the tube containing the fresh and dry flies, respectively.Under each developmental temperature (15, 20, 25, and 30 • C), we also tested the longevity of the adult offspring produced by each species.To do this, we monitored a group of newly emerged adult offspring (10 males+10 females in one food vial) weekly until the death of the final individual.In each trial, flies were transferred into a new food vial every week and 10 replicates were tested.

Desiccation and starvation resistance assays
In addition to the precedent life history traits, we also examined the survival capacities of the adult offspring when confronted with prolonged periods of severe dryness (desiccation) and food deprivation (starvation).We evaluated desiccation resistance using the methodology outlined by Chung and Carroll [6].For each developmental temperature and species, we exposed 10 adult offspring (7 days old) of the same sex to an assay setup (see Wang et al. [8]) containing 10 g of silica gel (S7500-1 KG, Sigma-Aldrich, Darmstadt, Germany).We recorded mortality every 2 h until the last individual succumbed.For the starvation resistance assays, we followed the   protocol described by Nayak and Mishra [32].Here, for each developmental temperature and species, 10 adult offspring (7 days old) of the same sex were removed from the food vial and transferred to a tube containing a moistened cotton plug at the bottom (to prevent death by desiccation).The opening was closed with a dry cotton plug.Mortalities were recorded daily until the death of the last individual.For each developmental temperature and species, we ran 10 independent trials in both desiccation and starvation resistance experiments.We performed all the experiments at 23 • C and 70 % humidity.

Data analysis
All analyses were conducted in R version 4.3.1 (R Development Core Team, 2023 [33]) and the Paleontological Statistics (PAST) version 3.12 [34] software.To visualize the divergence of the CHCs produced by each species across the different temperatures, we used the nonmetric multidimensional scaling (NMDS) with the Bray-Curtis dissimilarity distance and the analysis of similarity (ANOSIM) test [35].To compare the normalized intensity of each species' most abundant CHCs across the different developmental temperatures and between sex, we ran the two-way analysis of variance (ANOVA) followed by the Student-Newman-Keul (SNK) posthoc tests, using the R package called "agricolae" [36].We used the Chi-square test to compare the proportion of mated flies across the rearing temperature and species.To analyse mating duration in relation to rearing temperature and species, we conducted a two-way ANOVA followed by SNK posthoc tests.We used the same test to see whether the fertility, developmental time and body weight (fresh and dry) data varied across the different developmental times and species.We executed Kaplan-Meier survival analysis utilizing functions from the "survival" R package [37], including survfit() and survdiff(), in addition to the pairwise_survdiff() function available in the "survminer" R package (Kassambra et al., 2021).This analysis was conducted to examine variations in the survival of three drosophilid species concerning different rearing temperatures and species, following exposure to either desiccation or food deprivation.Also, the same analysis was used to compare the longevity data across the different rearing temperatures and species.To analyse the median lethal time data from the desiccation and starvation resistance bioassays as well as the longevity assays, we employed a two-way ANOVA, subsequently conducting SNK posthoc tests.Before running the ANOVA test, the normality (Shapiro test: P>0.05) and the homoscedasticity (Bartlett test: P>0.05) assumptions were verified.Statistical results were considered significant when P < 0.05.

Higher rearing temperatures lead to reduced mating
Having shown that increased rearing temperatures significantly affect cuticular hydrocarbon compositions in D. ezoana, D. novamexicana and D. virilis, we next aimed to see whether this would affect the mating behaviour of these flies, as some of the CHCs are known to mediate sexual communication [10].We found a significant change in both mating percentage (D.C again showed a higher mating percentage as compared to those developed at 15 and 30 • C (Fig. 3C, left).Here also, D. virilis couples developed at 30 • C mated for a shorter time (Fig. 3C, right).When we compared the mating percentage across the species, we observed that in contrast to D. novamexicana and D. virilis, D. ezoana showed a higher propensity to mate when developed at 15 and 20 • C than when they had developed at 25 and 30 • C (Supplementary Fig. 1A).We also observed that at both 15 and 30 • C, D. novamexicana had a shorter mating duration (Supplementary Fig. 1B).

Developmental temperature affects life history traits
After establishing the impact of rearing temperatures on the mating behaviour of the three drosophilid species, our next objective was to explore whether the treatments also influenced the fitness of the ensuing offspring.We first assessed the ability of the flies to produce viable adult offspring, a measure of fertility.Across the three species, we observed significant variations in fertility corresponding to changes in rearing temperature (Fig. 4A).Specifically, in D. ezoana (Fig. 4A  (Fig. 4B).
When adults emerged from these pupae, we observed a significant decrease in their fresh (Fig. 5C-supplementary table 1) and dry (Fig. 5D-supplementary table 2) body weights as developmental temperatures increased.The fresh body mass of adult offspring from D. ezoana (Fig. 5C We also observed that the longevity of adult offspring from D. ezoana (Fig. 5E, left), D. novamexicana (Fig. 5E, middle), and D. virilis (Fig. 5E, right) decreased significantly with increase of developmental temperatures.In the three species, adult offspring developed at 15, 20 and 25 • C lived longer than those developed at 30 • C. Specifically, in the case of D. ezoana (Fig. 5F, left; F 3-36 = 91.05,P<0.0001), D. novamexicana (Fig. 5F, middle; F 3-36 = 39.16,P<0.0001), and D. virilis (Fig. 5F, right; F 3-36 = 69.07,P<0.0001), offspring developed at 15 • C had higher median lethal time (LT50) than to those raised at 30 • C. Furthermore, when comparing across species, we observed that adult offspring of D. novamexicana in general had a shorter longevity than those of D. ezoana and D. virilis (Supplementary Fig. E and F).

Changes in rearing temperature affect resistance to desiccation and starvation
As in nature, a rise in temperature occasions water loss leading to the reduction of food availability [38], we assessed the desiccation and starvation resistance in the adult offspring of the three drosophilid species as developed at 15, 20, 25 and 30 • C. The results from the desiccation resistance experiment highlighted the significant impact of rearing temperature on the ability of the adult offspring from these species to endure and survive prolonged dry conditions (Fig. 6).In the case of D. ezoana, adult offspring developed at 20 • C and 25 • C exhibited markedly higher resistance to silica gel exposure compared to those raised at 15 • C and 30 • C (Fig. 6A, left), with higher median lethal time (LT50) (Fig. 6A

Discussion
Variations in rearing temperatures influence the composition of cuticular hydrocarbons (CHCs) in three Drosophila species: D. ezoana, found in Arctic regions; D. novamexicana, native to warm, desert environments and D. virilis, with a cosmopolitan distribution.Temperature changes in parallel affect mating behaviour, fertility, and other life-history traits in all three species, and also influence the desiccation and starvation resistance of the offspring.

CHC profiles and thermal adaptation
We observed distinct variations in profiles of cuticular hydrocarbon (CHCs) profiles among adult flies of the three drosophilids species when developed at varying temperatures.In the cold-adapted D. ezoana, we observed a consistent decline in the quantity of all CHCs as the developmental temperature increased.Such a trend was not observed in D. novamexicana and D. virilis (Fig. 2).For instance, D. novamexicana individuals developed at 15 and 20 • C produced lower amounts of (Z)-9-tricosene and 2-methyl triacontane compared to those developed at 25 and 30 • C.However, at the same temperature (15 and 20 • C), they synthesized higher amounts of 1heptacosanol and nonacosanal.Similarly, in D. virilis, adults developed at 15 and 20 • C produced higher quantities of 2-methyl tetracosane, (Z)-pentacosene, (1)-heptacosanol, and (Z)-13-docosen-ol, while displaying lower levels of 2-methyl-octacosane and nonacosanal.These observations highlight that temperature-dependent shifts in CHC profiles are species-specific.In fact, numerous  studies conducted at both the intra-and interspecific levels in insects have consistently confirmed the presence of this plasticity in CHC production in response to environmental stress [39][40][41].Furthermore, comparative studies across various drosophilid species have revealed that species from warmer climates tend to produce a higher proportion of methyl-branched CHCs, in contrast to those from cooler regions.In our case, we observed that, in addition to the common 2-methyl-octadecane produced by all three drosophilid species, the desert species, D. novamexicana and the cosmopolitan species D. virilis produced 2-methyl-triacontane and 2-methyl-tetracosane, respectively.Wang et al. [8] found a similar trend in Drosophila mojavensis, a desert species known for producing more methyl-branched CHCs.This phenomenon is not unique to Drosophila flies, as other desert insect species such as the stink beetle Eleodes armata [42], the locust Schistocerca gregaria [43], the ants Cataglyphis niger [44], and Pogonomyrmex barbatus [45] also exhibit high proportions of methyl-branched CHCs.Also, some grasshopper and ant species produced more n-alkanes in warmer climates [46,47].Wang et al. [48] discovered that the ability of D. mojavensis to synthesize these long methyl-branched cuticular hydrocarbons is linked to genetic variations in a fatty acyl-CoA elongase gene (mElo).However, it is important to note that their findings were based on D. mojavensis developed at room temperature.Conducting additional studies using flies developed at different temperatures (as we did) to assess the expression levels of the mElo gene will provide a deeper understanding of how the temperatures experienced during preimaginal development influence CHCs production in flies.

Mating success
As well as the significant changes in the compositions of CHCs, we observed alterations in the mating behaviour of the three drosophilid species in response to temperature variations during their development.In all three species, individuals developed at 15 • C and 30 • C exhibited lower mating success compared to those developed at 20 • C and 25 • C.This difference can be attributed to the impact of temperature on behaviour, morphology, and physiology [49,50].Insects that develop at extreme temperatures often experience adverse alterations in their biology, rendering them less competent in successful copulation.Lower temperatures tend to slow down metabolic processes, leading to reduced activity levels and increased time spent seeking warmth or shelter, which in turn limits their opportunities to mate [51].Conversely, insects developing at higher temperatures exhibit higher metabolic rates, which can make them more agitated and stressed, potentially disrupting their mating behaviours.Extreme temperatures can also influence the production, release, and detection of pheromones, affecting mate location and successful copulation.For example, henicos-1-ene (male specific pheromone) and (Z)-12-pentacosene from D. ezoana and D. virilis respectively are produced in lower amounts by individuals developed at 30 • C that exhibit lower mating success.Conversely, insects developed at more optimal temperatures tend to produce pheromones in quantities sufficient for successful mating.For instance, in D. novamexicana, individuals developed at 25 • C that show higher mating percentage also produce significantly higher amounts of (Z)-9-tricosene.
When comparing across species, we observed that the mating success of D. ezoana as compared to that of D. novamexicana and D. virilis was higher when they developed at 15 and 20 • C.However, developing at 25 and 30 • C, this species showed a lower mating percentage.This implies that the cold-adapted species (D. ezoana) may have evolved temperature-dependent reproductive traits that are more effective in colder conditions.Conversely, these traits evolved under warmer conditions in the warm-adapted (D. novamexicana) and the cosmopolitan (D. virilis) species.In D. melanogaster heat resistant flies mate more at elevated temperatures than heat-susceptible ones [52].Using 12 species of Drosophila species, Schnebel and Grossfield [53] observed that changes in temperature-dependent mating reflect differences in their thermal adaptation and geographic distributions.Moreover, high temperatures may decrease pheromonal CHC levels, leading to lower mating success.However, we observed an exception with D. virilis, where there was a significant decrease in (Z)-12-Pentacosene at 25 • C compared to 20 • C, yet the mating success percentage remained similar at both temperatures.One possible explanation is that mating in D. virilis depends not only on pheromones but also on courtship songs [54].
In addition to the observed changes in the mating success of the three drosophilid species, we also noted a substantial reduction in mating duration with the rise in developmental temperature.This change is likely a result of various factors, including changes in developmental speed, metabolic rates, behaviour and environmental conditions.Collectively, these factors contribute to shorter mating durations in insects exposed to higher temperatures during their development.The three drosophilid species had shorter developmental times (Fig. 5A) and lifespan (Fig. 5E and F) when developed at higher temperatures.Under such a scenario, insects may have less time available for mating before they reach the end of their natural lifespan [55].Furthermore, the increased metabolic activities in insects when developed at higher temperatures can result in quicker exhaustion of energy reserves, reducing the duration of mating activities [56].Moreover, insects developed at higher temperatures are perpetually exposed to heat and desiccation stress and tend to allocate fewer resources to reproduction and invest more in behavioural and physiological strategies aimed at bolstering their resilience to environmental stressors [57].This negative relationship between temperature and mating duration has also been observed in studies involving different insect species, such as the Japanese beetle Popillia japonica, bugs including Anthocoris tomentocus, A. whitei, A. nemoralis; [58], and the beetle Callosobruchus chinensis.

Fertility and life-history traits
We observed significant changes in fertility among the three drosophilid species in response to variations in their developmental temperatures.Specifically, individuals reared at 30 • C and 15 • C exhibited reduced production of adult offspring compared to those developed at 20 • C and 25 • C.These findings align with Cohet and David [59] who demonstrated that D. melanogaster females raised at various temperatures (ranging from 12 • C to 32 • C) produced more adult offspring when reared at 21 • C and 25 • C. Similar outcomes were also evident in Drosophila suzikii.Our study further revealed that flies reared at 15 • C and 30 • C displayed a decreased mating percentage, providing a potential explanation for the reduced adult offspring at these extreme temperatures.Additionally, extreme temperature conditions can adversely affect sperm production and viability in male insects, leading to diminished fertility due to lower fertilization success.For example, Gandara and Drummond-Barbosa [60] observed that D. melanogaster males reared at 29 • C produced fewer and lower-quality sperm, resulting in less efficient egg fertilization.This loss of fertility after exposure to high temperatures plays an important role in shaping the global distribution of several Drosophila species [61,62].Ovariole number emerged as another critical factor directly impacting fertility.Klepsatel et al. [63] found that D. melanogaster females developed at 17 • C and 29 • C exhibited lower ovariole numbers and displayed decreased fertility compared to those reared at 25 • C.Moreover, insect eggs are highly sensitive to both high and low temperatures, which can disrupt their development, reduce hatchability and result in the production of non-viable offspring [64].Among the three species, D. novamexicana produced fewer adult offspring.Notably, D. novamexicana females displayed a lower fresh body weight in comparison to D. ezoana and D. virilis females (see Supplementary Fig. 2C).According to Berger et al. [65], female body size is a reliable predictor of fertility in general.Larger female insects can accumulate more resources and convert them into eggs, often yielding more viable adult offspring.This positive correlation between body size and fertility is also found in various insect species including the moth Streblote panda [66], the beetle Callosobruchus chinensis [67,68], and the flies Drosophila malerkotliana and D. bipectinate [69].
We observed that as the developmental temperature increased, the females of the three drosophilid species produced adult offspring characterized by shorter development periods, decreased pupal and adult weights (both fresh and dry) and reduced longevity.Indeed, insect development is governed by enzymes whose activities increase in warmer conditions, consequently accelerating crucial chemical reactions essential for growth, moulting, and overall development [70,71].The observed reduction in size in our study can be attributed to accelerated development at higher temperatures, which offers the flies less time for feeding, growth, and nutrient storage before their pupation and emergence [72].Like in many ectotherms, our three drosophilid species follow the temperature-size rule (TSR) stating that in insects, an increase in developmental temperature leads to a decrease in final adult size [73,74].Using D. melanogaster, Li and Gong [75] demonstrated that development under cold conditions increases the production of insulin-like peptides leading to an increase in body size.Furthermore, apart from the reduction in body size, our study also uncovered a decrease in the lifespan of adult offspring produced by these three drosophilid species.A study using D. melanogaster from different genetic backgrounds showed that as temperature increases, the metabolic rate of insects rises, leading to higher energy expenditure and faster depletion of stored resources, thereby shortening their lifespan [76].Additionally, elevated temperatures can raise the production of detrimental reactive oxygen species (ROS) within the insect's body, potentially causing damage to cellular components and leading to oxidative stress, which is linked to both ageing and diminished longevity [77,78].
When compared at the species level, D. novamexicana exhibited smaller body size and decreased longevity in comparison to D. ezoana and D. virilis (Supplementary Fig. 2).In many insects including moths [79], cockroaches [80], and mosquitoes [81][82][83], smaller individuals tend to have shorter lifespans.This phenomenon can be attributed to the fact that smaller insects tend to be less efficient at accumulating food reserves and typically exhibit higher metabolic rates.Consequently, these factors can lead to accelerated ageing and a subsequent reduction in their overall lifespan [84].

Desiccation and starvation resistance
We found intra and interspecific variations of desiccation resistance in the three drosophilid species when developed at different temperatures.The flies exhibited lower resistance to water loss when developed at 15 • C. Adult insects emerging from development at lower temperatures, consistently exhibit increased susceptibility to desiccation stress due to reduced prior exposure to dehydration stress (Bong et al., 2021a;[85]).On the other hand, non-lethal exposure to desiccation stress during development increases resistance to subsequent exposures [86].This might explain why, when developed at 25 and 30 • C, the flies showed high desiccation resistance.When compared across species, the cold-adapted species (D. ezoana), was less resistant to water loss compared to the warm-adapted (D. novamexicana) and cosmopolitan (D. virilis) species.In fact, xeric-adapted and cosmopolitan insects like D. novamexicana and D. virilis respectively are generally more resistant to desiccation compared with mesic-adapted insects like D. ezoana ([87]; Matzkin et al., 2009a).This is mainly because they produce a higher number of methyl-branched CHCs as we previously elucidated (Fig. 2).Indeed there is a causal association between CHCs and desiccation survival [88], where an increase in desiccation resistance is associated with a high CHCs proportion [89].
We also observed changes in starvation resistance in response to variation in developmental temperature.In D. melanogaster, exposure to low temperatures during preimaginal development diminishes resistance to starvation, whereas higher temperatures enhance it [90].However, this was not the case in our three species.In particular, the cold-adapted D. ezoana and, to some extent, the cosmopolitan D. virilis exhibited heightened starvation resistance when developed at 15 • C (Fig. 7A and C).Conversely, individuals from the warm-adapted species, D. novamexicana (Fig. 7B), displayed reduced resistance to starvation when subjected to the same developmental temperature.When developed at 30 • C, D. ezoana experienced a significant reduction in starvation resistance compared to D. novamexicana and D. virilis.Matzkin et al. [91] observed that cactophilic drosophilid species, adapted to arid or desert regions, tend to withstand longer periods of food deprivation than their fruit-breeding counterparts.Insects in arid or desert regions often experience prolonged periods of food scarcity.They have evolved physiological and behavioral adaptations to reduce metabolic rates, optimize water use, and store energy more efficiently to survive extended periods of starvation [92].This provides a plausible explanation why D. novamexicana exhibited the highest starvation resistance.Additionally, earlier research by Da Lage et al. [93] unveiled that Afrotropical D. melanogaster flies, which inhabit hot environments, are approximately twice as resistant to starvation as their temperate counterparts, providing further insight into the complex relationship between developmental conditions and starvation resistance in drosophilid species.

Conclusion
We studied the impact of rearing temperature on three Drosophila species-D.ezoana, D. novamexicana, and D. virilis-across various facets, including cuticular hydrocarbon profiles, mating behaviour, fertility, life history traits, and desiccation and starvation resistance.Notably, each species exhibited unique variations in cuticular hydrocarbon profiles in response to different developmental temperatures, a phenomenon consistent with the notion that species from warmer climates tend to produce more methyl-branched CHCs.Mating behaviour was significantly affected by developmental temperature, with 15 • C and 30 • C leading to lower mating success, potentially attributed to alterations in behaviour, physiology and pheromone production.Furthermore, flies developed at 15 • C and 30 • C displayed reduced fertility, influenced by factors such as mating success and duration.The study also revealed that higher temperatures resulted in smaller body sizes and decreased lifespans in adult offspring, driven by accelerated development.In terms of resistance to desiccation and starvation, the warm-adapted and cosmopolitan species demonstrated greater resistance due to their higher proportion of cuticular hydrocarbons in comparison to the cold-adapted species.Despite all three species having experienced long breeding under laboratory conditions, potentially fostering experimental evolution, however, our findings suggest that various species with different thermal adaptations will indeed be affected by the global rise in temperature, particularly concerning their physiological chemistry and behaviours.Taken together, our study underscores the intricate relationship between developmental temperature, ecological adaptation and various life history traits, contributing valuable insights into how changes in environmental factors shape the biology and ecology of distinct species.

Fig. 2 .
Fig. 2. The cuticular hydrocarbon (CHC) composition of the three Drosophila species change in function of their rearing temperature.(A) D. ezoana CHC composition when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (B) D. novamexicana CHC composition when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (C) D. virilis CHC composition when developed at 15 • C, 20 • C, 25 • C, and 30 • C. On each figure panel, we present GC-MS chromatogram from different body wash samples, Non-Metric Multidimensional Scaling plot grouping body wash samples based on the rearing temperature, and boxplots illustrating the variation in normalized intensity of the most predominant CHCs across the body wash samples.In each boxplot, the ends of boxplot whiskers represent the minimum and maximum values of all the data and dots show individual data points.

Fig. 3 .
Fig. 3. Rearing temperatures affect the mating behavior of the three Drosophila species.(A) Left, bar graphs showing the variation of mating success in D. ezoana when reared at 15 • C, 20 • C, 25 • C, and 30 • C. Right, violin plots illustrating the change of the mating time of D. ezoana across the different rearing temperature.(B) Left, bar graphs depicting the variation of mating success in D. novamexicana when developed at 15 • C, 20 • C, 25 • C, and 30 • C. Right, violin plots illustrating the change of the mating time of D. novamexicana across the different rearing temperature.(C) Left, bar graphs depicting the variation of mating success in D. virilis when developed at 15 • C, 20 • C, 25 • C, and 30 • C. Right, violin plots illustrating the change of the mating time of D. virilis across the different rearing temperatures.Dots on each violin plot indicate data points from each replicate.Distinct letters on each graph denote significant differences (Mating percentage: Chi-square test; Mating duration: ANOVA followed by the SNK posthoc tests).

Fig. 4 .
Fig. 4. Rearing temperatures influence the fertility of the three Drosophila species.(A) Bar graphs showing the mean number of offspring produced by females of D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed and maintained at 15 • C, 20 • C, 25 • C, and 30 • C. (B) Bar graphs displaying the mean number of offspring produced by females of D. ezoana, D. novamexicana and D. virilis for each rearing temperature.Dots on each bar graph show individual data point.Error bars indicate standard error of the mean (SEM).Bars with different letters are significantly different from each other (ANOVA followed by the SNK posthoc tests; P < 0.05, n = 20-40).

Fig. 5 .
Fig. 5. Rearing temperatures affect the life history parameters of the offspring produced by the three Drosophila species.(A) Bar graphs showing the mean developmental time of the offspring of D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. Error bars indicate the standard error of the mean (SEM).Bars with different letters are significantly different from each other (ANOVA followed by the SNK posthoc tests; P < 0.05).(B) Boxplots depicting the pupae weight of D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (C) Boxplots depicting the fresh adult (female and male) body weight of D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (D) Boxplots depicting the dry adult (female and male) body weight of D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (E) Kaplan-Meier curves showing the variation of the longevity of adult offspring produced by D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. (F) Boxplots illustrating the change of the median lethal time of adult offspring produced by D. ezoana (left), D. novamexicana (middle) and D. virilis (right) when developed at 15 • C, 20 • C, 25 • C, and 30 • C. In each boxplot, the ends of boxplot whiskers represent the minimum and maximum values of all the data and dots show individual data points.Significant differences among the different temperature treatments are depicted by different letters (ANOVA followed by the SNK posthoc tests; P < 0.05, n = 10).

Fig. 6 .
Fig. 6.Rearing temperatures influence the desiccation resistance of the offspring produced by the three Drosophila species.(A-C) Kaplan-Meier curves (left) and Boxplots (right) showing respectively the change of the survivorship (after silica gel exposure) and median lethal time of the offspring of D. ezoana (A), D. novamexicana (B) and D. virilis (C) across the different rearing temperatures (15 • C, 20 • C, 25 • C, and 30 • C). (D-G) Kaplan-Meier curves (left) and Boxplots (right) illustrating respectively the variation of the survivorship (after silica gel exposure) and the median lethal time across the three Drosophila species when developed at 15 • C (D), 20 • C (E), 25 • C (F), and 30 • C (G).Each boxplot displays the median and whiskers indicate ±1.5 interquartile range limits.Dots on each box plot show individual data point.Box plots with different letters are significantly different from each other (ANOVA followed by the SNK posthoc tests; P < 0.05, n = 10).

Fig. 7 .
Fig. 7. Rearing temperature influence the starvation resistance of the offspring produced by the three Drosophila species.(A-B) Kaplan-Meier curves (left) and Boxplots (right) showing respectively the change of the survivorship (after food deprivation) and median lethal time of the offspring of D. ezoana (A), D. novamexicana (B) and D. virilis (C) across the different developmental temperatures (15 • C, 20 • C, 25 • C, and 30 • C). (D-G) Kaplan-Meier curves (left) and Boxplots (right) illustrating respectively the variation of the survivorship (after food deprivation) and the median lethal time across the three Drosophila species when developed at 15 • C (D), 20 • C (E), 25 • C (F), and 30 • C (G).In each boxplot, the ends of boxplot whiskers represent the minimum and maximum values of all the data and dots show individual data points.Box plots with different letters are significantly different from each other (ANOVA followed by the SNK posthoc tests; P < 0.05, n = 10).